PROJECT SUMMARY Circadian regulation, or 24-hour oscillations in behavior and biological function, is a conserved feature of biology that is present in diverse organisms from plants to vertebrates. While a widely-observed feature of aging is loss of circadian regulation, whether circadian-regulated processes are major factors that drive aging remains unknown. Loss of circadian regulation is typically thought to be pathological. In humans, loss of circadian regulation is associated with higher rates of obesity, diabetes, and infection. In this proposal, we present evidence from the fly model Drosophila melanogaster that a specific type of circadian dysregulation is not pathological but delays aging and extends lifespan. Because studies in Drosophila have provided crucial information about circadian regulation of biological functions such as immunity and metabolism, findings that have been replicated in vertebrates, Drosophila is well established as a useful model system. The core circadian clock consists of transcriptional activators and transcriptional inhibitors that cause 24-hour oscillations in gene expression and biological functions in both single cells and multicellular organisms. We found that loss of the conserved transcriptional inhibitors Timeless (Tim) or Period (Per) in Drosophila significantly extends lifespan by a mechanism independent of canonical longevity pathways such as insulin signaling, but due to upregulation of a conserved metabolic process called mitochondrial uncoupling via increased expression of the uncoupling protein UCP4C in the intestine. Our studies further implicate an anti- aging mechanism of lowering ROS levels and suppressing age-related stem cell expansion in the intestine. These findings suggest novel connections between circadian regulation, mitochondrial metabolism, stem cell biology, and lifespan. In this proposal, we will use the powerful genetic and cell biological methods in Drosophila to investigate the mechanisms by which circadian regulation modulates animal lifespan on the cellular, tissue- specific, and organism levels. The highly-conserved nature of both circadian regulation and this fundamental aspect of cellular metabolism, mitochondrial uncoupling, raise the exciting possibility that these mechanisms underlying lifespan extension are conserved in vertebrates. We will investigate the mechanisms by which loss of Per-mediated circadian regulation and upregulated mitochondrial uncoupling lead to lifespan extension in three ways: 1) we will determine the cellular and molecular mechanisms underlying circadian dysregulation and mitochondrial uncoupling that suppress age-related intestinal stem cell expansion; 2) we will determine whether mitochondrial uncoupling in the gut impacts the aging of other tissues; and 3) we will determine the functional window and other tissue-specific locations in which circadian dysregulation and mitochondrial uncoupling extend organism lifespan. As both circadian regulation and aging are regulated by evolutionarily conserved molecular mechanisms, we expect our work will have relevance for human health.